Richard I. Murahata

Suppressor T cells and suppressor macrophages induced by Corynebacterium parvum

Abstract Corynebacterium parvum, injected intravenously into C57B1/6 mice (H-2 b ) previously alloimmunized with P815 (H-2 d ) mastocytoma cells, generated splenic suppressor cells that inhibited the development of primary cytotoxic lymphocytes in vitro . These suppressor cells differed from those generated by intravenous C. parvum injection of naive C57B1/6 mice. The former suppressor cells were effectively induced by administration of 700 μg of C. parvum whereas the latter suppressor cells were dependent upon higher doses (1400 μg) of adjuvant for their activation. Furthermore, suppressor cells generated in alloimmunized mice could only suppress C57B1/6 anti-P815 in vitro cytotoxic responses whereas suppressor cells generated in naive mice could suppress C57B1/6 anti-CBA (H-2 k ) responses as well. Suppressor cells were not H-2 restricted in their action. Fractionation of spleens from alloimmunized, C. parvum -treated mice revealed the presence of suppressor T cells and suppressor macrophages. We were unable, however, to determine which cell was responsible for “antigen specificity” of suppression since the fractionation procedures seemed to trigger both suppressor cell types prior to adding them to the primary culture.

Partial purification and characterization of pemphigus-like antigens in urine.

SummaryThe inhibition of indirect immunofluorescence was used to detect the presence of pemphigus-like antigens in urinary proteins. Antigenic material was present in 12 of 40 urine samples obtained from 16 patients with bullous pemphigoid, cicatricial pemphigoid, or pemphigus vulgaris. Antigen could not be detected in the urine of patients with carcinoma of the bladder. Partial purification was accomplished by ion-exchange chromatography and column gel filtration. Antigenic activity was detectable in all fractions eluted from Sephadex G-200 in the broad range of molecular weight of 90,000–180,000 daltons. Pemphigus-like antigens were glycoproteins with an apparent molecular weight of 75,000 daltons by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). The antigens were immunogenic in mice, but were not detectable by double diffusion in agarose gels.

Activation and mechanism of action of suppressor macrophages.

Abstract Intravenous administration of Corynebacterium parvum to alloimmunized mice activates splenic suppressor macrophages that effectively curtail primary and secondary generation of cytotoxic T lymphocytes (CTLs) in vitro . CTL generation was significantly inhibited in suppressed primary cultures by Day 3, the earliest time point that activity is first detected in control cultures. Suppressor macrophages had to be present during the first 24–48 hr of culture to effectively curtail the generation of CTLs. However, if suppressor macrophages were reactivated by 48-hr in vitro culture and then added to primary sensitizations that had been initiated 48 hr previously, they were capable of significant suppression. Suppressor cells produced a soluble factor that mediated the inhibition of CTL generation. The production or action of this factor could not be counteracted by indomethacin.

Inhibition of memory cell-mediated cytotoxic response by systemic administration of Corynebacterium parvum

Abstract Intravenous administration of Corynebacterium parvum to mice during a developing immune response to alloantigens resulted in the marked inhibition of the generation and expression of memory cell-mediated cytotoxic response in the spleen. The inhibition was observed following rechallenge in vivo or by in vitro culturing with the same alloantigen. The impairment in vitro was due, in part, to the generation of regulatory cells which were non-T phagocytic cells, probably macrophages activated by C. parvum administration. These suppressor macrophages appear to act by inhibiting proliferation and clonal expansion of memory cytotoxic cells.

Detection of anti-intercellular cement substance and anti-basement membrane zone antibodies by radioimmunoassay using SCaBER tumour cell line as substrate.

IgG autoantibodies present in the serum of pemphigus vulgaris and bullous pemphigoid patients were detected by a solid phase radioimmunoassay, using a squamous cell tumour line, SCaBER, as substrate. This preliminary study shows that the SCaBER cell line displays both pemphigus and bullous pemphigoid antigens. This source of antigens should allow the development of a sensitive assay to measure anti‐ICS and anti‐BMZ antibodies. Such an assay may have clinical applications and may provide an important tool for studying the mechanisms of autoantibody production in pemphigus and pemphigoid.

Inhibition of cell-mediated cytotoxicity by Corynebacterium parvum

SummaryWe investigated the effect of altering dose and route of Corynebacterium parvum (C. parvum) administration on the adjuvants inhibition of cell-mediated cytotoxicity (CMC). Primary in vivo and secondary in vitro CMC of C57B1/6 mice alloimmunized to P815 were depressed if C. parvum was administered systemically (IV or IP) but not when it was given SC. Similarly, only systemic C. parvum generated cells capable of suppressing in vitro CMC. Primary and secondary CMC in spleen was equally inhibited by 700 and 70 μg, whereas suppressor cell activity was marked with 700 μg and minimal with 70 μg. Administration of C. parvum SC admixed with alloantigen resulted in early enhancement and late depression of primary CMC. Secondary CMC was depressed but suppressor activity was absent. Dissociation of CMC depression from suppressor cell generation indicates that these phenomena can be separated under certain conditions.

Dissociation of biological activities of Corynebacterium parvum by chemical fractionation

Fractionation of Corynebacterium parvum (C. parvum) by phenol-water extraction resulted in components which differentially affected the cell-mediated cytotoxic response to tumor alloantigens in mice. The residue of the extraction (Fraction C) and its light component (Fraction D) retained all of the activities of the whole micro-organism, i.e. inhibition of primary spleen cell cytotoxicity, inhibition of in vitro memory cell-mediated cytotoxicity (CMC) and generation of macrophage-like suppressor cells. Administration of the phenol phase extracted material (Fraction A) inhibited both the primary and memory cytotoxic responses, but did not elicit suppressor macrophages. Destruction of carbohydrate components of Fraction D significantly reduced its ability to inhibit the memory response and to elicit suppressor macrophages. In contrast, its ability to inhibit the primary cytotoxic reponse was unaffected. This study clearly demonstrates that the several effects of C. parvum administration on the cell-mediated cytotoxic response to tumor alloantigens can be associated with different components of the organism. Furthermore, the dissociation of the effects indicate that the generation of primary cytotoxic lymphocytes, the priming of memory cytotoxic lymphocytes, and the induction of regulatory macrophages are not necessarily linked during the generation of a normal cytotoxic response.

Systemic administration of corynebacterium parvum during sensitization to tumor alloantigen-modified response to rechallenge.

SummaryIntravenous administration of Corynebacterium parvum (C. parvum) to mice during a primary immune response against tumor alloantigens impairs their ability to generate memory cell-mediated cytotoxicity (CMC) in response to an intraperitoneal rechallenge with the same tumor alloantigens. Decreased CMC was observed in spleen and mesenteric lymph nodes, whereas CMC of lymphoid populations from the peritoneal cavity was merely delayed, reaching comparable levels to those found in control animals by day 5. Serum levels of cytotoxic antibody were unaffected, indicating that C. parvum administered during a primary immune response has selective effects on the cytotoxic memory response.